Gene That Controls Parthenocarpic Fruit Formation and Uses Thereof

The present disclosure relates to a gene that controls parthenocarpic fruit formation and uses thereof.

In general, in angiosperms, fruits can develop normally only after pollination and fertilization. However, in some angiosperms, a phenomenon occurs in which the ovary develops into a fruit without fertilization and the fruit is seedless. This phenomenon is called parthenocarpy.

Naturally, parthenocarpy occurs because, in bananas and tangerines, fertilization does not occur because the pollen does not develop, even though pollination occurs; in pineapples, fertilization does not occur due to self-incompatibility, even though pollination occurs; in pears and apples, the temperature is excessively low during the flowering period; and in persimmons, the plant is aged.

Although fruit development by pollination is greatly affected by the environment, fruit development by parthenocarpy has the advantage of being relatively less affected by the environment. For example, tomato mutants that form fruits even at high or low temperatures or at low light intensity have been reported. As another example, the fruit of parthenocarpic pepper has been reported to have a low susceptibility to blossom-end rot. In addition, due to their seedless characteristics, parthenocarpic crops such as parthenocarpic watermelon, grapes, and bananas have the advantage of being easier and more convenient to consume, which can increase marketability.

Due to these advantages, there are cases where parthenocarpy is artificially induced in the field of agriculture or horticulture. For example, there are known cases of crossbreeding pepper and ground cherry, which are distantly related species that cannot be fertilized, and methods of stabbing the ovary of tobacco with a platinum wire. More commonly, there are cases of treating the stigma of apple or watermelon with auxin, and cases of spraying gibberellin on grapes.

Auxin and gibberellin, plant hormones used to induce parthenocarpy, have been widely reported to be involved in fruit formation, although their exact roles are unknown. When either auxin or gibberellin acts on unfertilized ovules, fruit development occurs in many plant species. Because of their role in fruit setting, these hormones are also used in agriculture to improve fruit setting.

Under this background, the inventors of the present disclosure have identified a novel gene controlling parthenocarpy, Solyc04g072920, and developed, based on transformation, a plant capable of producing parthenocarpic fruits without separate mechanical or chemical treatment, and a method for producing the same.

Therefore, an object of the present disclosure is to provide a composition for inducing parthenocarpy in a plant, comprising, as an active ingredient, a SlTPP4 gene expression inhibitor, a SlTPP4 protein activity inhibitor, or a SlTPP4 gene mutagen, wherein the SlTPP4 protein comprises the amino acid sequence of SEQ ID NO: 2, and the SlTPP4 gene encodes the STPP4 protein.

Another object of the present disclosure is to provide a method for inducing parthenocarpy in a plant, comprising a step of inducing a loss-of-function mutation in a gene encoding a SlTPP4 protein comprising the amino acid sequence of SEQ ID NO: 2, inhibiting the expression of the gene, or inhibiting the activity of the SlTPP4 protein.

Still another object of the present disclosure is to provide a transgenic plant or plant part exhibiting a parthenocarpic phenotype by the method for inducing parthenocarpy in a plant.

The present disclosure relates to a gene that controls parthenocarpic fruit formation and uses thereof.

Hereinafter, the present disclosure is described in more detail.

One aspect of the present disclosure is a composition for inducing parthenocarpy in a plant, comprising, as an active ingredient, a SlTPP4 gene expression inhibitor, a SlTPP4 protein activity inhibitor, or a SlTPP4 gene mutagen, wherein the SlTPP4 protein comprises the amino acid sequence of SEQ ID NO: 2, and the SlTPP4 gene encodes the SlTPP4 protein. The SlTPP4 gene may comprise the nucleotide sequence of SEQ ID NO: 1, without being limited thereto.

As used herein, the term “parthenocarpy” refers to a phenomenon in which an ovary develops into a fruit without fertilization and the fruit is seedless. When the composition of the present disclosure is used to induce a loss-of-function mutation in the SlTPP4 gene of a plant or inhibit the expression of the gene, facultative parthenocarpy is induced. “Induction of facultative parthenocarpy” means that whether or not parthenocarpic fruit is formed varies depending on whether or not pollination occurs. That is, the term means that seedless parthenocarpic fruits are produced when no pollination occurs, and normal seeded fruits are produced when pollination occurs.

The nucleotide sequence of SEQ ID NO: 1 is the SlTPP4 gene located on chromosome 4 of tomato (), which encodes trehalose-6-phosphate phosphatase that is 365aa in length. The SlTPP4 gene was after solyc04g072920, which was confirmed to be one of the eight SlTPPs of tomato, and has been reported to be mainly expressed in the roots.

The present inventors have found that, when interference or loss-of-function mutation of the SlTPP4 gene in a tomato plant is induced, the tomato plant exhibits parthenocarpic characteristics, which means that seedless fruits develop when the flowers are not pollinated. On the other hand, even loss-of-function mutants of the SlTPP4 gene can develop normal seeded fruits when flowers are pollinated. This type of parthenocarpy is called facultative parthenocarpy because it appears only in the absence of pollination. Therefore, the SlTPP4 gene is responsible for the parthenocarpic phenotype in plants such as tomato, and enables control of parthenocarpy. The SlTPP4 loss-of-function mutant in the example of the present disclosure produces seedless parthenocarpic fruits from unpollinated flowers, and produces normal seeded fruits from pollinated flowers.

The SlTPP4 gene expression inhibitor may include, in the broadest sense, all substances that can inhibit SlTPP4 gene expression by acting on one or more steps of the SlTPP4 protein expression process. Specifically, the SlTPP4 gene expression inhibitor may be selected from the group consisting of an antisense nucleotide, small interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, DNAyzme, and peptide nucleic acids (PNAs), which bind complementarily to a sequence of the SlTPP4 gene, a sequence complementary to the sequence of the SlTPP4 gene, or a fragment of the mRNA sequence of the SlTPP4 gene, thereby inhibiting expression of the SlTPP4 gene, without being limited thereto.

The antisense nucleotide binds (hybridizes) to a complementary nucleotide sequence of DNA, immature mRNA. or mature mRNA, as defined by Watson-Crick base pairing, thereby interrupting the flow of genetic information from DNA to protein.

The siRNA may be composed of a sense sequence selected from the mRNA nucleotide sequence of the gene encoding the SlTPP4 protein, and an antisense sequence complementary to the sense sequence. The sense sequence may be 15 to 30 mer in length, without being limited thereto.

The SlTPP4 protein activity inhibitor may be selected from the group consisting of a compound, a peptide, a peptide mimetic, a substrate analog, an aptamer, and an antibody, which bind to the SlTPP4 protein, without being limited thereto.

The compound includes any compound that may bind specifically to the SlTPP4 protein and inhibit the activity thereof.

The aptamer is a single-stranded DNA or RNA molecule, and may be obtained by isolating an oligomer, which binds to a specific chemical molecule or biological molecule with high affinity and selectivity, by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). The aptamer may bind specifically to a target and regulate the activity of the target. For example, the aptamer may block the function of the target by binding to the target

The SlTPP4 gene mutagen may be either a complex (ribonucleoprotein) of a guide RNA specific to the target nucleotide sequence of the SlTPP4 gene and an endonuclease protein, or a recombinant vector comprising a DNA encoding a guide RNA specific to the target nucleotide sequence of the SlTPP4 gene and a nucleic acid sequence encoding an endonuclease protein.

As used herein, the term “target gene” means a portion of DNA within the genome of a plant in which a mutation is to be induced through the present disclosure. The type of target gene is not limited, and the target gene may include both a coding region and a non-coding region. A person skilled in the art may select the target gene according to the desired mutation for the plant to be produced, depending on the purpose.

As used herein, the term “guide RNA” refers to RNA specific for DNA encoding a nucleotide sequence of a target gene, and refers to ribonucleic acid that complementarily binds to all or part of a target DNA nucleotide sequence and directs an endonuclease protein to the target DNA nucleotide sequence. The guide RNA refers to a dual RNA comprising two RNAS, namely, crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as components, or refers to a single-stranded guide RNA (sgRNA) comprising a first region including a sequence that is completely or partially complementary to a nucleotide sequence in the target gene and a second region including a sequence that interacts with an RNA-guided nuclease. However, as long as the RNA-guided nuclease is in a form that can be active in the target nucleotide sequence, any guide RNA may be included in the scope of the present disclosure without limitation, and may be appropriately selected according to techniques known in the art in consideration of the type of endonuclease used therewith or the microorganism from which the endonuclease is derived.

In addition, the guide RNA may be, but is not limited to, a guide RNA transcribed from a plasmid template, a guide RNA transcribed in vitro (e.g., an oligonucleotide double-strand), or a synthetic guide RNA.

In the composition for inducing parthenocarpy in a plant according to the present disclosure, the guide RNA is designed to be specific to the target nucleotide sequence of the SlTPP4 gene. For example, the target nucleotide sequence of the SlTPP4 gene may be composed of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, without being limited thereto. The nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 is the sequence of the first or second region where the PAM sequence (NGG) is located within the ORF of the SlTPP4 gene consisting of the nucleotide sequence of SEQ ID NO: 1.

In the composition for inducing parthenocarpy in a plant according to the present disclosure, the endonuclease protein may be at least one selected from the group consisting of Cas9, Cpf1 (CRISPR from Prevotella and

Francisella 1), TALEN (transcription activator-like effector nuclease), ZEN (zinc finger nuclease), or functional analogs thereof, and preferably may be a Cas9 protein, without being limited thereto.

In addition, the Cas9 protein may be at least one selected from the group consisting of a Cas9 protein derived from, a Cas9 protein derived from, a Cas9 protein derived fromor, a Cas9 protein derived from, a Cas9 protein derived from, a Cas9 protein derived from, and the like, without being limited thereto. The Cas9 protein or its genetic information thereof may be obtained from known databases such as GenBank of NCBI (National Center for Biotechnology Information).

The Cas9 protein is an RNA-guided DNA endonuclease enzyme that induces double-stranded DNA breaks. In order for the Cas9 protein to bind precisely to the target nucleotide sequence and cut the DNA strand, a short sequence of three nucleotides known as a Protospacer-Adjacent Motif (PAM) should be present next to the target nucleotide sequence, and the Cas9 protein forms a cleavage between the 3and 4base pairs from the PAM sequence (NGG).

In the composition for inducing parthenocarpy in a plant according to the present disclosure, the guide RNA and the endonuclease protein may form a ribonucleoprotein complex and may function as an RNA-guided engineered nuclease (RGEN).

Another object of the present disclosure is a method for inducing parthenocarpy in a plant, comprising a step of inducing a loss-of-function mutation in a gene encoding a SlTPP4 protein comprising the amino acid sequence of SEQ ID NO: 2, or inhibiting the expression of the gene, or inhibiting the activity of the SlTPP4 protein.

The inducing of the loss-of-function mutation may be achieved by introducing into a cell of the plant either a complex (ribonucleoprotein) of a guide RNA specific to a target nucleotide sequence of the SlTPP4 gene and an endonuclease protein, or a recombinant vector comprising a DNA encoding a guide RNA specific to a target nucleotide sequence of the SlTPP4 gene and a nucleic acid sequence encoding an endonuclease protein, without being limited thereto.

The guide RNA specific to the target nucleotide sequence of the SlTPP4 gene and the endonuclease protein are as described above.

The CRISPR/Cas9 system used in the present disclosure is a gene editing method using the non-homologous end joining (NHEJ) mechanism that introduces a double-strand break at a specific location of a specific gene to be edited, thereby inducing an insertion-deletion (InDel) mutation by incomplete repair induced during the DNA repair process.

In the method for inducing parthenocarpy in a plant according to the present disclosure, the method for transducing the complex of the guide RNA and the endonuclease protein into plant cells may be suitably selected from a calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, (DNA or RNA-coated) particle bombardment of various plant materials, infection with (non-integrative) viruses inmediated gene transfer, and the like.

In addition, introducing into a cell of the plant the recombinant vector comprising the DNA encoding the guide RNA specific to the target nucleotide sequence and the nucleic acid sequence encoding the endonuclease protein is referred to as a transformation method. Transformation of plant species is now routine for plant species, including both the Dicotyledoneae as well as the Monocotyledoneae. In principle, any transformation method may be used to introduce a recombinant vector according to the present disclosure into a suitable ancestor cell.

The “plant cell” into which the guide RNA specific to the target nucleotide sequence and the endonuclease protein are to be introduced may be any plant cell. The plant cell is a cultured cell, a cultured tissue, a cultured organ, or a whole plant. “Plant tissue” includes differentiated or undifferentiated plant tissues, for example, but not limited to, root, stem, leaf, pollen, seed, cancerous tissue, and various types of cells that are used for culture, i.e., single cell, protoplast, shoot, and callus tissue. The plant tissue may be in planta or in organ culture, tissue culture, or cell culture.

In the method for inducing parthenocarpy in a plant according to the present disclosure, any method known in the art may be used to regenerate the plant with the loss-of-function mutation induced. Genome-edited plant cells should be regenerated into a whole plant. Techniques for the regeneration of mature plants from callus or protoplast culture are well known in the art for a large number of different species.

The inhibition of expression of the gene may be achieved by treatment with an SlTPP4 gene expression inhibitor selected from the group consisting of an antisense nucleotide, small interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, DNAyzme, and peptide nucleic acids (PNAs), which bind complementarily to the mRNA sequence of the SlTPP4 gene, a sequence complementary to the mRNA sequence of the SlTPP4 gene, or a fragment of the mRNA sequence of the SlTPP4 gene, thereby inhibiting the expression of the SlTPP4 gene, without being limited thereto.

Another aspect of the present disclosure is a transgenic plant or plant part exhibiting a parthenocarpic phenotype by the method for inducing parthenocarpy in a plant.

In a method according to one embodiment of the present disclosure, the plant may be a food crop selected from the group consisting of rice, wheat, barley, corn, soybean, potato, wheat, red bean, oat, and sorghum; a vegetable crop selected from the group consisting of Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; a special crop selected from the group consisting of ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanut, and rapeseed; a fruit tree selected from the group consisting of apple trees, pear trees, jujube trees, peaches, kiwis, grapes, citrus fruits, persimmons, plums, apricots, and bananas; a flowering plant selected from the group consisting of roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, and tulips; or a forage crop selected from the group consisting of ryegrass, red clover, orchardgrass, alfalfa, tall fescue, and perennial ryegrass.

Preferably, the plant may be a dicotyledonous plant such as tomato, Arabidopsis, potato, eggplant, tobacco, pepper, burdock, crown daisy, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, water parsley, parsley, Chinese cabbage, cabbage, mustard greens, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, or pea. More preferably, the plant is tomato.

The plant part may be a fruit, a flower, a leaf, a stem, a cutting, an ovule, pollen, a root, a seed, a rhizome, a scion, an embryo, anther, a cell, or a protoplast.

The plant may contain a mutant of SlTPP4 gene represented by a nucleotide sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 11, without being limited thereto.

The present disclosure relates to a gene that controls parthenocarpy and uses thereof. Specifically, when the expression of the gene of the present disclosure is inhibited or a loss-of-function mutation in the gene is induced, it is possible to produce a plant capable of forming parthenocarpic fruits. The parthenocarpic plant produced by the present disclosure exhibits facultative parthenocarpy, and thus may form seedless parthenocarpic fruits when pollination does not occur, and may produce seeded fruits, like those in general plants, when pollination occurs.

The parthenocarpic fruit of the present disclosure exhibits a fruit formation rate of up to 90% of that of a wild-type pollinated fruit, and also a fresh weight of 70% of that of the wild-type pollinated fruit. In addition, it was confirmed that it is possible to form parthenocarpic fruits even under high-temperature stress conditions in which a wild-type plant cannot form pollinated fruits.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these examples are intended to illustrate one or more embodiments, and the scope of the present disclosure is not limited to these examples.

Micro-Tom, a tomato (L.) cultivar, was used in the present disclosure. Seeds were placed on the rockwool cube (40×40×40 mm; Grodan, Roermond, Netherlands) and cultivated with a tomato-specific nutrient solution (Daema Inc. Korea), with an electrical conductivity (EC) of 2.2 dS m-1 under a photoperiod of 16-hr light/8-hr dark at a light intensity of 150 μmol msand a temperature of 25° C.